Normal Myeloid Progenitors Research Articles

Abstract 5092: Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors

Abstract Clinical success of antigen-directed therapies in cancer has been limited by heterogeneous antigen expression and a narrow therapeutic window. In many patients, failure of antigen-directed therapies such as CAR T-cell therapies, antibody-drug conjugates (ADC) or radioligand therapies (RLT) has less to do with a lack of potency or specificity of the agent and more to do with variable and suboptimal antigen expression in the patient. We are developing combination therapies that introduce a priming agent designed to optimize antigen expression in the patient and improve the therapeutic window of existing antigen-directed therapies. Our high-throughput platform utilizes machine learning and automation to scale functional combinatorial screening for small molecule priming agents using cell lines and patient samples. Using this platform, we are identifying priming therapies for CD123, CD33, CD22, and CD19 in hematological malignancies including acute myeloid leukemia (AML), and HER2, HER3, and TROP2 in solid tumors including breast cancer.For our CD123 program, we have identified priming agents that selectively upregulate CD123 expression ≥2-fold on AML cells without increasing expression on normal myeloid progenitor cells, which also express CD123. AML cells treated with a CD123 priming agent in combination with a CD123 ADC showed greater tumor-cell killing compared to the ADC alone. In comparison, normal myeloid progenitor cells from healthy bone marrow treated with the same CD123 priming-ADC combination treatment did not show an increase in ADC-mediated cell killing. From our mechanistic studies, we have identified a somatic mutation associated with CD123 upregulation that may lead to selective upregulation on cancer cells and not on normal cells. Additionally, this marker can be used for patient selection. We have also observed CD123 upregulation in vivo using mouse models of AML and are currently conducting additional in vivo proof of concept studies to demonstrate that CD123 priming therapy enhances efficacy.There are two distinct advantages of a priming approach paired with an antigen-directed therapy, which include: (1) improving the durability of patient responses and (2) expanding the patient population. Our priming combination approach has the potential to overcome antigen heterogeneity and thereby improve the clinical success of antigen-directed therapies. Citation Format: Kathryn E. Vanderlaag, Anukriti Dhar, Molly Volkman, Erik Werner, Transon Nguyen, Kamran Ali. Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5092.

CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric acute myleoid leukemia (AML) remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the major histocompatibility complex-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial (clinicaltrials gov. Identifier: NCT01371981). Thirty-eight percent of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower white blood cells and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event-free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro preclinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AML at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

AML Immunotherapy Using a Novel Tcrm-Based Bispecific Antibody That Targets a Leader Sequence Peptide Derived from Cathepsin G

Introduction: Myeloid azurophil granules provide a rich source of intracellular leukemia antigens. Cathepsin G (CG) is a serine protease that has higher expression in AML blasts in comparison to normal myeloid progenitors. Based on the unique biology of HLA-A*0201 (HLA-A2), in which presentation of leader sequence (LS)-derived peptides is favored, we focused on the LS-CG-derived peptide CG1 (FLLPTGAEA). LS peptides are naturally processed and loaded on HLA-A2, providing an abundant source of surface peptide/HLA (pHLA) targets. We eluted CG1 from the surface of primary HLA-A2 + AML blasts and AML cell lines, and demonstrated CG1-targeting immunity in leukemia patients following allogeneic stem cell transplant, hence highlighting the potential for CG1 to be a promising immunotherapeutic target in AML. Numerous platforms have been developed to target cell surface pHLA complexes. T cell receptor (TCR) mimic (m) antibodies are immunotherapeutic antibodies that target pHLA, which are the natural ligands for the TCR. TCRm antibodies have been a breakthrough in immunotherapy, as they: (1) target intracellular antigens expressed on HLA; (2) are engineered to have high affinity for HLA; (3) can elicit their cytotoxic effect independent of the TCR; and (4) can be produced in large quantities providing an off-the shelf-product for patient use. Here we report on the engineering, preclinical efficacy, and safety evaluation of a novel CG1-targeting, T cell-engager, bispecific antibody (CG1xCD3e), which incorporates a novel TCRm construct that recognizes cell surface CG1/HLA-A2 complexes. Methods: To generate the CG1/HLA-A2-binding monoclonal antibodies, H2L2 human transgenic mice were immunized with CG1/HLA-A2 monomers subcutaneously. The resulting paired heavy and light chains were cloned from the antigen-sorted memory B cells into the single pcDNA3.1(+) vector with both human (h) immunoglobulin (Ig)G1 and hKappa for subsequent ExpiCHO high-throughput transient expression. Antibody binding was assessed by Bio-Layer Interferometry (BLI) and T2 cellular binding assays. Xencor anti-CD3e scFv and Knobs-in-holes technology was applied for the bispecific antibody engineering for xMab format. In vitro activity of CG1xCD3e and T cell cytokine secretion was confirmed using flow cytometry-based T cell-dependent cellular cytotoxicity (TDCC). The specificity of CG1xCD3e for leukemia was assessed using colony forming unit assays. In vivo efficacy was evaluated in NSG mice engrafted with AML cell lines and treated with CG1xCD3e with normal donor peripheral blood mononuclear cells (ND-PBMC). Results: Using BLI, binding avidity of the CG1xCD3e to CD3-Fc, KD= 7.52 x 10-10 M, and affinity to CG1/HLA-A2, KD= 1.28 x 10-10 M were calculated. Using flow cytometry, we confirmed high affinity binding of CG1xCD3e to CG1/HLA on the CG1- expressing EM2 HLA-A2 + cell line, and also demonstrated binding of CG1xCD3e to CD3 using jurkat T cell line. After co-culturing GFP/Luciferase + HLA-A2 + AML (U937-A2 and ML-2) or CML (EM2) cell lines with ND-PBMC and different concentrations of CG1xCD3e bispecific antibody for 24 or 48 hours, flow cytometry analysis demonstrated highly specific CG1/HLA-A2 dependent killing of AML cells by T cells cultured with CG1xCD3e bispecific antibody, in comparison with control bispecific antibody with a disabled CG1 binding arm. This correlated with both tumor- and bispecific antibody- dependent T cell activation and cytokine secretion. To study the in vivo anti-leukemia activity of CG1xCD3e, we engrafted NSG mice with the HLA-A2 + ML-2 and U937-A2 AML cell lines, and treated mice after tumor engraftment with ND-PBMC (1 x 107 cells) and weekly intraperitoneal injections of CG1xCD3e bispecific antibody (0.01 mg/Kg, 0.05 mg/Kg, and 0.1 mg/Kg) or PBS. Bioluminescence imaging demonstrated that the mice treated with ND-PBMC + CG1xCD3e had significantly lower levels of leukemia burden compared to the mice treated with ND-PBMC alone (Figure 1). Lastly CFU assays using HLA-A2 + normal donor marrow in a semi-solid matrix of methylcellulose in the presence of T cells and either CG1xCD3e bispecific antibody or isotype antibody, confirmed specificity of CG1xCD3e bispecific antibody for leukemia. Conclusion: Our study provides strong pre-clinical evidence supporting the targeting of LS- derived peptides, specifically CG1, in the setting of AML using a novel TCRm-based bispecific antibody.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Late B Cell-Specific Dis3-Knockout Mice Do Not Develop Plasma Cell Neoplasm

Recent advances in next-generation sequencing have unveiled genetic abnormalities associated with multiple myeloma (MM). Of note, DIS3 mutations have been observed in ~10% of MM patients, and 13q deletion including the DIS3 gene locus are present in ~40% of MM patients. Furthermore, DIS3 mutations/deletions have been shown to be associated with poorer prognosis in MM. Regardless of high incidence of DIS3 mutations/deletions and their relation to adverse outcome, the roles of DIS3 in hematopoiesis and myelomagenesis remains incompletely understood. Here we show that Dis3 is required for normal hematopoiesis and Dis3 deficiency is not sufficient for the development of plasma cell neoplasm in mice. We first explored DIS3 functions in hematopoiesis by generating Dis3 conditional knockout (KO) mice (Dis3 fl/fl;Mx-Cre). To avoid possible effects of Dis3 deficiency on nonhematopoietic cells, we transplanted bone marrow (BM) cells from Dis3 fl/fl;Mx-Cre mice into lethally irradiated CD45.1 + recipient mice. Four weeks after transplantation, we knocked out Dis3 by inducing Cre recombinase via an intraperitoneal injection of Poly(I:C). The deletion of Dis3 in BM cells was confirmed by genotyping and quantitative real time PCR. Importantly, loss of Dis3 in hematopoietic cells resulted in severe pancytopenia at 2 weeks after Dis3 deletion. Consistent with this observation, Dis3 KO mice showed reduced BM counts with relative preservation of mature lymphocytes. A flow cytometric analysis of the BM exhibited lower frequencies of Lin -Sca-2 +c-Kit + (LSK), hematopoietic stem, and multipotent progenitor cells in Dis3 KO mice compared with wild-type (WT) mice. Dis3 KO mice also exhibited lower frequencies of common myeloid progenitors, granulocyte/macrophage progenitors, and megakaryocyte/erythroid progenitors compared with WT mice. A frequency of common lymphoid progenitors (CLPs) was comparable between WT and Dis3 KO mice, whereas absolute numbers of CLPs were significantly reduced in Dis3 KO mice than in WT mice. These results indicate that Dis3 is indispensable for normal hematopoiesis, and LSK and myeloid progenitor cells are more dependent on Dis3 than lymphoid progenitor cells. We next examined whether loss-of-function of Dis3 drives plasma cell neoplasm. To do so, we crossed Dis3 fl/wt or Dis3 fl/fl mice with Cgamma1-Cre mice and generated late B cell-specific Dis3 KO mice by activating Cre recombinase via immunizing with NP-CGG in these compound mice. However, these mice did not develop any B cell malignancies and shorten the survival compared with WT mice. In the samples of MM patients, the frequency of DIS3 mutations is significantly higher in t(14;16) samples than in those without t(14;16) (Walker et al. Blood 2018). We therefore utilized t(14;16) model mice (c-Maf TG) (Morito et al. Cancer Res 2011) and generated c-Maf TG; Dis3 fl/wt;Cgamma1-Cre and c-Maf TG; Dis3 flfl;Cgamma1-Cre mice. As reported (Morito et al. Cancer Res 2011), a small fraction of mice with c-Maf TG developed B cell lymphoma after a long latency. However, the addition of Dis3 KO did not increase the frequency of B cell lymphoma and shorten the survival compared with c-MAF TG mice. These results indicate that only Dis3 deficiency and the combination of c-Maf overexpression and Dis3 deficiency do not develop plasma cell neoplasm and suggest that additional oncogenic events are necessary for myelomagenesis. Further investigations are required for elucidating the mechanisms of how loss-of-function of DIS3 is involved in the development of MM.

Selective activation of STAT3 and STAT5 dictates the fate of myeloid progenitor cells

The molecular programs that govern the directed differentiation of myeloid progenitor cells are still poorly defined. Using a previously established immortalized, phenotypically normal myeloid progenitor cell model mEB8-ER, we unveil a new mechanism mediated by STAT5 and STAT3 at a bifurcation point of myeloid progenitor cell-fate specification. We find that myeloid progenitor cells can spontaneously differentiate into neutrophils with a basal level of STAT3 phosphorylation, which is enhanced by G-CSF treatment or STAT3 over-expression, leading to elevated neutrophil differentiation. Reduced STAT3 phosphorylation caused by GM-CSF treatment, STAT3 specific inhibitor, or STAT3 depletion leads to attenuated myeloid differentiation into neutrophils, while elevating differentiation into monocytes/macrophages. In contrast, STAT5 appears to have an antagonistic function to STAT3. When activated by GM-CSF, STAT5 promotes myeloid differentiation into monocytes/macrophages but inhibits neutrophil differentiation. At the mechanistic level, GM-CSF activates STAT5 to up-regulate SOCS3, which attenuates STAT3 phosphorylation and consequently neutrophil differentiation, while enhancing monocyte/macrophage differentiation. Furthermore, inhibition of STAT5 and STAT3 in primary myeloid progenitors recapitulates the results from the mEB8-ER model. Together, our findings provide new mechanistic insights into myeloid differentiation and may prove useful for the diagnosis and treatment of diseases related to abnormal myeloid differentiation.

Abstract 1548: CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells

Abstract The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short, and overcoming therapeutic resistance has proven difficult. The presence of chemoresistant leukemic stem cells (LSCs), among other mechanisms, has been implicated as a driver in the observed relapse. Antibody-drug conjugates (ADC) are targeted cancer therapeutics that leverage antigen specificity to deliver a potent cytotoxic payload. Recently, ADCs have shown promise in the clinic, but can also present safety and efficacy concerns. Mylotarg, an FDA approved CD33-directed ADC used to treat Acute Myeloid Leukemia, was the first ADC to be approved. The therapeutic is active against most AML tumors but can lead to severe myelotoxicity given the expression of CD33 on normal myeloid progenitor cells. We leveraged a pharmaco-pheno-multiomic analysis of primary AML tumors to uncover a previously unexplored target expressed on both LSCs and AML blasts, but not normal hematopoetic progenitor cells or other normal tissue. The target also showed capacity to rapidly internalize and has activity of well-established oncogenic kinases associated with expression. A novel monoclonal antibody (IgG1) with favorable binding and biophysical characteristics was established against this novel target and conjugated to a highly potent cytotoxic agent, creating a novel ADC. This ADC shows strong binding across AML tumors with effective internalization and highly potent killing of AML tumors. Citation Format: Garima Kaushik, Bandana Vishwakarma, Marina Bell, Kakajan Komurov. CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1548.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.

Targeting Myeloid Leukemia Using Novel TCR-like Chimeric Antigen Receptor-T Cells Against Cathepsin G

Cathepsin G (CG) is a serine protease that is normally contained within primary granules of neutrophils and is expressed during the promyelocyte stage of myeloid cell development. We reported CG to be highly expressed in acute myeloid leukemia (AML) blasts compared with normal myeloid progenitors and leukocytes. We also showed CG to be a promising immunotherapeutic target in AML, having eluted a CG-derived peptide, FLLPTGAEA (hereafter referred to as CG1), from the surface of primary HLA-A*0201 (HLA-A2+) AML blasts and AML cell lines. Additionally, we showed CG expression in acute lymphoblastic leukemia (ALL) through endogenous expression by some ALL subsets and by cross-presentation from the marrow microenvironment. We also showed that CG1-specific cytotoxic T-lymphocytes (CTL) killed HLA-A2+ primary AML blasts and cell lines, but not normal myeloid progenitor cells. Furthermore, we detected CG1-CTL in peripheral blood mononuclear cells (PBMC) from AML patients following allogenic stem cell transplant. Here we report on the engineering, preclinical efficacy, and safety of CG1-targeting chimeric antigen receptor (CAR) T cells, which employ a novel T cell receptor-like (TCR-L) CAR construct that binds cell surface CG1/HLA-A2. Methods Time-of-flight mass spectrometry was used to quantify CG1 expression on the cell surface. The antibody targeting CG1/HLA-A2 was developed by immunizing Harbour Mice with CG1/HLA-A2 complex, FACS-sorting of single B cells, screening B cell antibody-containing supernatant for binding to CG1/HLA-A2, and fusion cloning of VH and VL sequences from positively screened B cells. OCTET BLI assays were used to determine the binding of different alanine substituted CG1 monomers to lead antibodies. The affinity of each monomer was calculated based on BLI data. T2 binding assays were used to validate the binding of antibody monomers to cell surface CG1/HLA-A2 complexes. Immunohistochemistry (IHC) using human fresh and frozen PBMC and marrow was used to show the specificity of anti-CG1/HLA-A2 antibodies. CG1 ScFV was cloned into pORBIT-LENTI-IRES-EGFR-III vector co-expressing either CD28 (CG1-CD28-CAR) or 4-1BB (CG1-41BB-CAR). HLA-A2+ normal donor (ND) T- cells were activated with anti-CD3 and anti-CD28 antibodies before lentiviral transduction with CG1 CAR constructs. Transduced cells were expanded and then characterized in vitro to determine CAR T quality. In vitro flow cytometry cytotoxicity assays were used to confirm in vitro killing of leukemia by CG1-CAR T cells. NSG mice engrafted with AML cell lines and AML patient-derived xenografts (PDX) were used to demonstrate the in vivo efficacy/pharmacokinetics/pharmacodynamics of CG1-CAR T cells from different donors. Results We eluted CG1 peptide from the cell surface in 7 of 8 HLA-A2+ myeloid leukemia cell lines. CG1 was absent on the cell surface of HLA-A2+ non-myeloid solid tumor and lymphoid cell lines, as well as in normal HLA-A2+ neutrophils and PBMC. Screening of different antibody clones for binding to CG1 peptide with different alanine amino acid substitutions identified top 5 antibody candidates with high affinity (single to sub-digit nM) for CG1-HLA-A2. T2 binding assays demonstrated high binding specificity of these five CG1 antibodies to CG1 monomers. The lead antibodies for CAR-T development were selected based on high antibody binding affinities (0.14-5 nM), specificity for CG1/HLA-A2, and no binding to ND marrow and PBMC. In vitro cytotoxicity assays confirmed in vitro killing of HLA-A2-transduced U937 (U937-A2) cells by CG1-CAR T cells. Both CG1-CD28- and the CG1-41BB-CAR constructs demonstrated high levels of killing. Lastly, in vivo AML mouse models using ML-2 and U937-A2 cell lines, and AML PDX validated the in vivo activity of CG1-CD28- and CG1-41BB-CAR T cells (Figure). Conclusion Targeting CG1 using immunotherapy appears to be a valid approach in the setting of AML. We first showed that CG was highly expressed and presented on AML blasts. We developed TCRL-scFv that targets CG1/HLA-A2 on the cell surface and generated two different CAR constructs based on the scFv antibody sequence. We then showed potent anti-leukemia activity and specificity of CG1-CAR T cells against human leukemia in vitro and in vivo. Together, these data provide the pre-clinical proof of concept work that lays the foundation for further development of CG1-CAR T cells as a potential immunotherapy for patients with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

C/EBPα-p30 Confers AML Cell Susceptibility to the Terminal Unfolded Protein Response and Resistance to Venetoclax By Activating DDIT3 Transcription

Introduction: CCAAT-enhancer-binding protein α (C/EBPα) is one of the most essential lineage-specific transcription factors involved in myeloid differentiation and includes two isoforms of protein: full-length C/EBPα-p42 and truncated C/EBPα-p30. Acute myeloid leukemia (AML) with CEBPA biallelic (CEBPAbi) mutation or single mutation in the bZIP region is associated with a favorable prognosis, but the mechanisms are still unclear. The unfolded protein response (UPR) is a conserved adaptive signal transduction pathway triggered by endoplasmic reticulum (ER) stress. UPR first strives to restore protein homeostasis and maintain cell survival through a pro-survival arm called adaptive UPR. However, in the setting of irremediable ER stress, the UPR transforms into pro-apoptotic terminal UPR signaling that promotes cell apoptosis. DNA damage-inducible transcript 3 (DDIT3) is a key transcription factor responsible for terminal UPR under severe ER stress. Here, we hypothesize that C/EBPα two isoforms regulate DDIT3 transcription, resulting in easier elimination of AML cells with CEBPA mutations and dysregulation by terminal UPR. Methods: Human AML cell lines and mouse 32Dcl3 were transduced with recombinant lentiviruses to knockdown CEBPA expression or over-express of C/EBPα two isoforms. RT-qPCR was used to detected the mRNA levels and western immunoblotting was used to determine the protein levels. The cell apoptosis rates were determined as the percentage of Annexin V-positive cells by flow cytometry. We performed CFU assays to confirm the declined granulocytic differentiation of 32Dcl3 cells with Cebpa knockdown. To identify potential C/EBPα-binding DNA sequences in the genome, we performed ChIP-seq. Luciferase reporter analysis was used to confirm the transcriptional regulation ability of C/EBPα two isoforms and independent ChIP-qPCR of candidate sequences were performed to further verify the transcription factor binding sites of C/EBPα. Finally, we generated a xenograft model with NCG mice to determine the potential application value of our findings. Results: Firstly, we found a negative correlation between CEBPA expression and DDIT3 levels in AML cell lines and primary de novo AML patients. After knockdown of CEBPA in THP-1 cells, which has high endogenous C/EBPα expression, DDIT3 expression was upregulated, and an increased apoptotic rate of AML cells was induced by ER stress. Cebpa knockdown also induced easier elimination of mouse myeloid progenitor cells 32Dcl3 due to upregulation of Ddit3, so as to avoid leukemogenesis since their differentiation was blocked. After the overexpression of C/EBPα-p42 and C/EBPα-p30 in NB4 cells, which have relatively low levels of endogenous C/EBPα isoforms, we found that C/EBPα-p30 activated DDIT3 expression to increase the sensitivity of AML cells to tunicamycin. However, C/EBPα-p42 had the opposite effect to C/EBPα-p30. Through luciferase reporter analysis, we confirmed that C/EBPα-p30 and C/EBPα-p42 directly regulate the transcription of DDIT3 in a contrasting manner, indicating that the two isoforms maintain protein homeostasis corporately. ChIP-seq and ChIP-qPCR revealed that C/EBPα-p30 has a special binding site in DDIT3 gene promoter with stronger affinity than C/EBPα-p42 (hg38, Chr12:57520654-57521026). Given that the BCL2 family proteins are major targets of DDIT3, we further observed that a low p42/p30 ratio was related to reduced BCL2 levels-a major target of DDIT3, increased MCL1 levels, and resistance of AML cells to venetocalx. Finally, in vitro and in vivo we found that tunicamycin and sorafenib could synergistically reverse venetoclax resistance in AML cells by activating the terminal UPR and inhibiting MCL1. Conclusions: Collectively, our results identified a critical regulation of C/EBPα two isoforms on the transcription of DDIT3 in AML cells as well as normal myeloid progenitor cells and confirmed the importance of this regulation in leukemia surveillance and elimination of AML cells. Moreover, we demonstrated that patients with AML having a low C/EBPα p42/p30 ratio and CEBPAbi mutation may not benefit from monotherapy with BCL2 inhibitors. This can be resolved by a combination of ER stress inducers or MCL1 inhibitors, including tunicamycin and sorafenib, which may represent a novel therapeutic strategy for AML patients with CEBPA mutations and dysregulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AML-147 C-MYC Targeting by Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Exerts Profound Cell Kill In Vitro and In Vivo in Acute Myeloid Leukemia and Lymphomas

The oncoprotein c-Myc governs epigenome and transcriptome and is deregulated in 70% of all human cancers. MYC is highly expressed in TP53 mutant or venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been met with success. PROTACs or cerebron E3 ligase modulators (CELMoDs) are attractive modalities to specifically target hitherto undruggable oncoproteins. We developed the first c-Myc degrader GT19630 (GT19715, the salt form of GT19630). We tested it in cell-free, cellular assays and in animal studies. GT19630 effectively degraded oncogenic c-Myc protein (IC50 = 1.5 nM) in HL-60 cells. C-Myc was effectively pulled down by biotinylated GT19630 in a cell-free, in vitro affinity purification assay; and a proteasome inhibitor ixazomib completely blocked c-Myc degradation. IC50 of GT19715 in HL-60 cells was 1.8 nM, being considerably lower than 40.2 nM, an IC50 of normal myeloid progenitors in CFU assay, suggesting a therapeutic window. GT19630 shares chemical properties with other CELMoDs and proteomic analyses revealed degradation of translation termination factor G1 to S phase transition proteins 1 (GSPT1), an important factor in LSC survival (Surka et al. Blood 2021). Indeed, GT19630 effectively degrades GSPT1 along with complete degradation of c-Myc in a xenograft model with HL-60 cells, and inhibits tumor growth at a dose as low as 0.3 mg/kg/bid. GT19630 had no effect on normal myeloid lineages in rats at 6 mg/kg. GT19715 eliminates circulating blasts and prolongs survival in the c-Myc-driven systemic Daudi leukemia/lymphoma model. Importantly, GT19715 induces cell killing independent of TP53 status, and baseline c-Myc protein levels significantly correlated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout or mutations of TP53 (R2 = 0.86, P = 0.02). We found that MV4;11 ven resistant (VR) cells demonstrated elevated protein levels of c-Myc, and GSPT1 and exhibited greater sensitivity to GT19715compared to ven-sensitive parental cells. Finally, GT19715 significantly reduced human CD45+ AML blasts compared to vehicle control in vivo in an AML PDX model. First results with the novel dual c-Myc/GSPT1 degrader GT19715 demonstrate promising preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development.

Identification of Leukemia-Associated Immunophenotypes by Databaseguided Flow Cytometry Provides a Highly Sensitive and Reproducible Strategy for the Study of Measurable Residual Disease in Acute Myeloblastic Leukemia.

Simple SummaryThe complete immunophenotypic characterization of acute myeloid leukemia is essential for an accurate diagnosis and follow-up, which is determinant in the course of the disease. In many cases, the only option for the evaluation of minimal residual disease is flow cytometry, so the aim of this study is to develop an automatized multidimensional strategy to identify and characterize LAIPs as well as to detect new emerging aberrances in AML patients during the follow-up. The integrated DFN/LAIP strategy that we propose allows the identification of the most useful markers for minimal residual disease monitoring, improving the sensitivity and specificity of these studies. Furthermore, the use of databases and the automation of the analysis provide the basis for the generation of objective conclusions in minimal residual disease evaluations.Background: Multiparametric Flow Cytometry (MFC) is an essential tool to study the involved cell lineages, the aberrant differentiation/maturation patterns and the expression of aberrant antigens in acute myeloid leukemia (AML). The characterization of leukemia-associated immunophenotypes (LAIPs) at the moment of diagnosis is critical to establish reproducible strategies for the study of measurable residual disease using MFC (MFC-MRD). Methods: In this study, we identify and characterize LAIPs by comparing the leukemic populations of 145 AML patients, using the EuroFlow AML/ MDS MFC panel, with six databases of normal myeloid progenitors (MPCs). Principal component analysis was used to identify and characterize the LAIPs, which were then used to generate individual profiles for MFC-MRD monitoring. Furthermore, we investigated the relationship between the expression patterns of LAIPs and the different subtypes of AML. The MFC-MRD study was performed by identifying residual AML populations that matched with the LAIPs at diagnosis. To further validate this approach, the presence of MRD was also assessed by qPCR (qPCR-MRD). Finally, we studied the association between MFC-MRD and progression-free survival (PFS). Results: The strategy used in this study allowed us to describe more than 300 different LAIPs and facilitated the association of specific phenotypes with certain subtypes of AML. The MFC-MRD monitoring based on LAIPs with good/strong specificity was applicable to virtually all patients and showed a good correlation with qPCR-MRD and PFS. Conclusions: The described methodology provides an objective method to identify and characterize LAIPs. Furthermore, it provides a theoretical basis to develop highly sensitive MFC-MRD strategies.

SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion.To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs.SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype.SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis.HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes.While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. DisclosuresNo relevant conflicts of interest to declare.

A Phase I/II Clinical Trial of Piggybac-Modified GMR CAR-T Cell Therapy for CD116 Positive Relapsed/Refractory Myeloid Malignancies

Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan.Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells.The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed.Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029).Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. [Display omitted] DisclosuresSaito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Abstract 505: Oral azacitidine modulates the immune microenvironment in acute myeloid leukemia (AML) patients in remission: A subanalysis from the QUAZAR AML-001 Trial

Abstract BACKGROUND The immunologic effects of maintenance therapy in patients (pts) with AML in remission are not well-characterized but of high clinical interest, as rapid recovery of bone marrow (BM) after intensive chemotherapy (IC) may help delay relapse. Post IC, immunological interactions in the BM microenvironment present several immunosuppressive mechanisms. PD-L1 is commonly overexpressed on AML blasts, which is associated with worse prognosis. Oral azacitidine (Oral-AZA [CC-486]) is a hypomethylating agent recently approved in the US for pts with AML in complete remission (CR) or CR with incomplete hematologic recovery (CRi). To better understand the effects of Oral-AZA on immune cells, checkpoint expression of PD-L1/2 on AML blasts and normal myeloid progenitors (MPs), and the kinetics of T cell recovery and activation/exhaustion (eg, PD1, TIM3) were assessed. METHODS Biomarker-evaluable pts aged ≥ 55 years with AML were randomized 1:1 to Oral-AZA 300 mg (n=56) or placebo (PBO, n=52) post IC within 4 months of achieving CR. Flow cytometry evaluations of BM aspirates were performed at screening (ie, baseline [BL]), every 3 cycles until cycle 24 and every 6 cycles thereafter to cycle 36, or as clinically indicated. Correlative analyses of baseline immune parameters with median (med) relapse-free survival (RFS) were computed using Kaplan-Meier methods. RESULTS In the biomarker-evaluable pts, PD-L1 and PD-L2 expression at BL were higher on AML blasts (med intensity 7.1 and 2.9) than normal MPs (0.7 and 1.6). Most AML blasts were PD-L2+ (79%), whereas only 1.9% were PD-L1+. When stratified by the med, higher BL CD3 T cell numbers (as a % of total BM ) were associated with favorable RFS in both Tx arms (Oral-AZA: ≥ med, 562 days[d] and < med, 235d [P = .0308]; PBO: ≥ med, 325d and < med, 155d [P = .0391]). At cycle 3, pts in the Oral-AZA arm had a 1.7-fold increase in CD3 T cells from BL (PBO, 1.1; P = .0450), suggesting Oral-AZA can promote immunologic recovery during early Tx cycles. There was an inverse correlation between T cell exhaustion marker phenotypes (PD1/TIM3+) with CD4 (r = -.5967; P < .0001) and CD8 (r = -.2484; P = .0095) T cell numbers. An increase in RFS was seen in the PBO arm with lower PD1/TIM3+ CD4 numbers (< med, 429d; ≥ med, 155d; P = .0037), with a nominal increase observed in the Oral-AZA arm (< med, 428d; ≥ med, 303d; P = .6764). In a subset of pts, Oral-AZA appeared to suppress CD4 T cell exhaustion (PD1/TIM3+) compared with PBO. CONCLUSIONS Pts in CR/CRi post-IC have a unique immune profile defined by high expression of PD-L1 on a subset of blasts and a high % of PD-L2+ blasts. A higher BL CD3 T cell count after IC in BM was prognostic. Additionally, Oral-AZA appears to contribute to an increase in T cells while also suppressing exhaustion, potentially promoting T cell signaling that could activate functional immune-mediated responses against residual leukemic cells. Citation Format: Daniel L. Menezes, Wendy L. See, Alberto Risueno, Jianglin Ma, Ignazia La Torre, Barry Skikne, CL Beach, Keshava Kumar, Anjan Thakurta. Oral azacitidine modulates the immune microenvironment in acute myeloid leukemia (AML) patients in remission: A subanalysis from the QUAZAR AML-001 Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 505.

CD99 Regulates Normal and Malignant Stem Cell Function By Restricting Protein Translation

We recently reported that CD99 is highly expressed on disease initiating stem cells in myeloid malignancies. In acute myeloid leukemia (AML), high expression of CD99 identified human leukemia stem cells (LSCs) with enhanced leukemia-initiating capacity and gene signatures associated with hematopoietic stem cell (HSC) self-renewal. LSCs with high CD99 expression were also marked by significant depletion of ribosomal protein transcripts, and we have subsequently confirmed that they exhibit reduced levels of protein translation as measured by polysome profiling.To elucidate the mechanisms by which CD99 promotes self-renewal in hematopoietic cells, we characterized CD99-deficient mice generated by gene trapping (B6- CD99Gt(pU-21T)44lmeg , or CD99 Gt/Gt). At steady-state, homozygous CD99 Gt/Gt mice exhibit normal CBCs, bone marrow (BM) cellularity, and hematopoietic stem and progenitor frequencies. To test the function of CD99 Gt/Gt HSCs, we FACS-purified HSCs (Lin-Sca-1+c-Kit+CD34-CD150+) from CD99 Gt/Gt and wild-type (WT) mice and transplanted them into lethally irradiated WT recipients. At early time points, CD99 Gt/Gt HSCs and WT HSCs engrafted equally well. However, over the course of 24 weeks, there emerged in mice transplanted with CD99 Gt/Gt HSCs a strong trend towards decreased peripheral blood (PB) donor chimerism, as well as a progressive and highly significant decline in granulocyte (Mac-1+Gr-1+) donor chimerism, suggesting that CD99 Gt/Gt HSCs may gradually exhaust after transplantation (Fig.1A). BM evaluation at 24 weeks revealed no differences in HSC donor chimerism, but there was a significant decrease in myeloid progenitor (Lin-Sca-1-c-Kit+) donor chimerism and BM cellularity in mice transplanted with CD99 Gt/Gt HSCs (Fig.1B), consistent with decreased HSC functional output. In non-competitive secondary transplants using unfractionated BM, CD99 Gt/Gt HSCs exhibited a marked decrease in engraftment, consistent with impaired self-renewal.To assess the impact of CD99-deficiency on transcriptional output, we performed RNA-seq on HSCs from CD99 Gt/Gt and WT mice, identifying 327 differentially expressed genes (p<0.01). Gene set enrichment analysis identified an increase in transcripts associated with the proteasome, mTOR signaling, and c-MYC targets, as well as depletion of ribosomal protein transcripts (Fig.1C). This gene expression pattern suggests that CD99 Gt/Gt HSCs exhibit proteotoxic stress, which has been shown to promote transcriptional repression of ribosomal proteins.Given that tightly regulated translation has been shown to be essential for HSC function, as well as our findings in HSCs and LSCs that suggest that CD99 negatively regulates translation, we hypothesized that CD99 promotes self-renewal in HSCs by limiting protein translation. To test this, we transplanted HSCs from CD99 Gt/Gt and WT mice as before and treated recipient mice with the mTOR inhibitor rapamycin (4 mg/kg daily). Rapamycin completely rescued the engraftment defect of CD99 Gt/Gt HSCs at eight weeks (Fig.1D), strongly suggesting that their functional defects are mediated by increased translation.Finally, we measured CD99 expression on leukemia cells from mouse AML models including MLL-AF9 , AML-ETO9a , and NUP98-HOXD13, findingCD99 to be overexpressed compared with normal myeloid progenitors (Lin-Sca-1-c-Kit+), similar to our prior observations in human AML. Because AML1-ETO is correlated with high CD99 mRNA expression in human AML (ECOG 1900 cohort), we tested whether CD99 is required for AML1-ETO induced leukemogenesis. c-Kit+ cells from CD99 Gt/Gt and WT mice were transduced with AML1-ETO 9a (AE9a) and transplanted into lethally irradiated recipients. While there was no significant difference in survival between mice transplanted with CD99 Gt/Gt-AE9a vs. WT-AE9a cells (81 d vs. 108 d, p=0.32), CD99 Gt/Gt-AE9a grafts exhibited less splenomegaly and a dramatic reduction in c-Kit+ cells (43-fold, p=0.0064), suggestive of increased differentiation of leukemia cells and depletion of LSCs (Fig.1E). Consistent with this, RNA-seq revealed in CD99 Gt/Gt-AE9a cells depletion of HSC/LSC-associated gene signatures, as well as enrichment for ribosomal protein transcripts (Fig.1F).Together, these findings reveal an essential role for CD99 in HSC and LSC function, suggesting that it promotes self-renewal in part by acting as a key regulator of protein translation. [Display omitted] DisclosuresLevine:Roche: Research Funding; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding. Park:Forty Seven, Inc.: Consultancy.

Chimeric Antigen Receptor (CAR) T Cell Therapy for CD7-Positive Acute Myeloid Leukemia

Expression of CD7 is an adverse prognostic factor in both AML and myelodysplastic syndromes. Because CD7 is not constitutively expressed by mature myeloid cells or early myeloid progenitor cells, we explored the feasibility of targeting these malignancies using T cells expressing a CD7-directed chimeric antigen receptor (CAR7). Due to high expression of CD7 on normal T cells, expression of CAR7 induced substantial fratricide of T cells, preventing their expansion and long term survival. We therefore disrupted the CD7 gene in T cells using CRISPR/Cas9 prior to transduction with the CAR. Electroporation of the Cas9 protein with CD7-specific gRNA produced little toxicity to T cells, and the resulting CD7KOCAR7 T cells (henceforth CAR7 T cells) had no evident fratricide, expanding >300-fold within two weeks.Upon coculture with primary AML blasts, CAR7 T cells eliminated a mean 85% of malignant cells within 48h (Fig. A) and expanded 5-fold. Furthermore, CAR7 T cells effectively targeted primitive AML progenitor cells resulting in a mean >25-fold decrease in the number of leukemic colonies after a 5h coculture (Fig. B). CAR7 T cells demonstrated robust cytotoxicity against CD7+ AML cell lines eliminating >95% of malignant cells after 72h of coculture at a 1:40 effector-to-target ratio but had no activity against CD7-negative tumor cells. To test the protective effect in vivo, we established a mouse xenograft model of disseminated AML by intravenous injection of the CD7+ AML cell line KG-1a. A single injection of 2x10^6 CAR7 T cells produced a >2-log reduction in tumor burden and significantly extended median survival (65 days in control group; median survival not reached at 88 days for the CAR7 T treated animals, P=0.0025). In a second, more aggressive AML model using Kasumi-3 cells, we observed a 2-log reduction in tumor volume following administration of CAR7 T cells.CD7 may also be transiently expressed in normal early myeloid progenitors. To assess the off-tumor hematotoxicity of CAR7 T cells, we cocultured them with cord blood-derived hematopoietic progenitor cells and measured colony formation on a methylcellulose medium. We observed no differences in the numbers of granulocytic, monocytic and erythrocytic colonies after exposure to CAR7 T or control non-transduced T cells indicating a lack of toxicity to normal myeloid and erythroid progenitor cells (Fig. C). Similarly, we observed no reactivity of CAR7 T cells against peripheral monocytes. However, CAR7 T cells were cytotoxic to normal peripheral T- and NK-cells suggesting potential aplasia of these lymphocyte subsets subsequent to in vivo administration of the CAR7 T cells. We may be able to circumvent this toxicity, by infusing pathogen-directed CD7KO T cells from the same donor to repopulate the host, since CD7KO cells appear to retain TCR-mediated activity against a range of pathogens. Hence, CD7KO T cells expressing a CD7-directed CAR can expand without fratricide and have high activity against AML while sparing normal myeloid cells. These data support the feasibility of this approach for the targeted therapy of AML. [Display omitted] DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees.

Acute Myeloid Leukemia Classification System That Associates Normal Myeloid-Cell Subset Gene Signatures with Prognosis

Background: Acute Myeloid Leukemia (AML) represents a group of heterogeneous clonal disorders of hematopoietic progenitor cells that are driven by multiple genetic events that impair cell differentiation and apoptosis and invoke uncontrolled cell proliferation. Consequently, immature myeloid blasts rapidly accumulate in the bone marrow and block the generation of mature end stage cells, which eventually causes potentially fatal infections and hemorrhages. AML is characterized by extensive genetic and phenotypic heterogeneity, which currently is diagnostically classified based on blast morphology defined by the French-American-British (FAB) Cooperative Group, cytogenetic abnormalities, and improved knowledge of genomic and epigenomic alterations. However, this has not yet resulted in treatment progress. AML patients are still routinely treated with a standard first-line combinatorial chemotherapy regimen with an overall survival rate remaining below 20%. Therefore, there is an unmet need for novel methods to stratify patients into therapy-relevant subtypes.Concept and Aims: We hypothesize that phenotypic heterogeneity of AML is a consequence of dysregulated differentiation and that classification based on transcriptional reminiscence of cell of origin (COO) during AML transformation is of pathogenetic impact and therefore provide diagnostic and prognostic value. It is the aim of this project to generate and validate a COO classification system based on normal myeloid-cell-subset-associated gene signatures (MAGS) to stratify AML patients into therapy-relevant and prognostic subtypes.MAGS Generation: Based on gene expression profiles of publicly available sorted normal myeloid subpopulations, we generated a COO classifier including the CD34+/CD38- hematopoietic stem cells (HSC), CD34+/CD38+/CD45RA- megakaryocyte-erythroid progenitors (MEP), and CD34+/CD38+/CD45RA+ granulocytic-monocytic progenitors (GMP) using regularized multinomial regression with 3 discrete outcomes and the elastic net penalty. The regularization parameters were chosen by cross validation.Results: MAGS prediction accuracy was validated in an independent dataset (n = 38; accuracy = 0.79) of sorted normal myeloid subpopulations. The prognostic value of MAGS assignment was studied by survival analyses using the Kaplan Meier method, log-rank test statistics, and simple and multiple Cox proportional hazards regression analysis in 2 clinical cohorts (TCGA: n = 115; GSE6891: n = 431). MAGS assignment had significant prognostic associations with overall survival (Figure 1; Table 1A) revealing that the linage committed MAGS subtypes GMP and MEP showed superior prognosis compared to the undifferentiated AMLs captured by the MAGS HSC subtype. Furthermore, multiple Cox regression analysis with MAGS assignment (HSC, GMP, MEP), FAB score (M0-M6), cytogenetics (good, intermediate, poor), and age (dichotomized into <55 and ≥55) as independent explanatory variables showed that MAGS assignment provides independent prognostic information (Table 1B), indicating that the MAGS classification captures distinct pathogenetic and prognostic knowledge not already explained by the FAB- or cytogenetic classifications. Molecular characterization of MAGS subtypes by differential gene expression analysis, gene set enrichment analysis, and identification of subtype specific mutation patterns of well-documented AML driver mutations indicated a reduced proliferation rate as well as overrepresentation of EVI1 mutations associated with the poor responding HSC subtype.Conclusion: In accordance with our previous reports in malignant B-cell disorders, we have documented a novel classification system that associates normal myeloid progenitor subsets with AML subtypes of distinct pathogenetic and prognostic importance not already explained by the FAB- or cytogenetic classifications. Further pathogenetic studies are needed to develop a diagnostic platform using MAGS-assigned subtypes to improve disease management. [Display omitted] DisclosuresSeverinsen:ARIAD: Research Funding; Celgene: Research Funding; Novartis: Research Funding. El-Galaly:Roche: Other: Travel funding; Takeda: Other: Travel funding.

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

Flow cytometry reveals the nuances of clonal haematopoiesis

Flow cytometry reveals the nuances of clonal haematopoiesis